HPLC is used ubiquitously for both analytical and preparative separation of molecules in diverse areas such as research, development and production in chemistry, pharmaceuticals, biotechnology, fundamental life science studies, etc.
One particular use of HPLC is in the field of proteomics, i.e. the study of the entire protein complement of a cell or tissue sample where proteolytic fragments of proteins (e.g. peptides) are separated by HPLC prior to detection by mass spectrometry. Since the samples being analyzed in proteomics experiments are typically very complex and available in only very low quantities, it is frequently a challenge to obtain sufficient sensitivity and analysis speed.
Sensitivity is obtained by using low flow rates for the mobile phase in combination with nano-bore columns (i.e. columns of narrow inner diameter). This approach however often leads to back pressures that are in excess of the instrumental system tolerances, which may cause system failure and also in many cases requires prolonged analysis times, which in turn leads to poor duty cycles for the overall LC-MS analysis. These back pressure and duty cycle problems affect many other application areas beyond proteomics as well.
In known proteomics, typical experimental HPLC conditions and parameters currently are:
Flow range: 100 nL/min-500 nL/min (also called nano-LC)
Pressure range: 50 atm.-600 atm.
Liquid phases are typically:
A-buffer: Mainly aqueous, often acidified and containing additives, with no or low organic content
B-buffer: Mainly organic, often acidified and containing additives, with no or low water content
Gradients are typically: From no or low percentage B-buffer to high percentage B-buffer in 5 to 600 minutes. Standard gradients could be from 5% B to 90% B in 30 minutes.
Stationary phases are typically:
Beads of various materials, often highly porous, diameters of 1.5 μm-5 μm, hydrophobic coating of hydrocarbons (C8 or C18) with chemical end-cap (non-hydrocarbon functional group)
Column sizes are typically:
ID: 25 μm to 250 μm
Length 1 cm to 200 cm
The operation of reverse phase nano-HPLC (or rather, the execution of an analysis cycle) can be separated into several chronologically distinct steps:                1. Loading of sample; either from an auto-sampler or by manual injection with a syringe into a sample loop. Sample volumes in nano-LC applications are typically 0.5 μL to 10 μL (but can be 10 times larger or smaller than this range).        2. Re-location of the loaded sample from the loop and onto the column.        3. De-salting of the immobilized sample by a volume of buffer (typically 100% A-buffer, i.e. with little or no organic solvent). This volume is typically 1.5 to 3 times larger than the volume from which the sample was loaded (e.g. a 5 μL sample injection would be de-salted with 7.5 μL to 15 μL of buffer) but the de-salting buffer volume can also be much larger or smaller than this range.        4. The elution and separation step. This is either done as isocratic elution (i.e. where the buffer composition remains constant during the elution step) or as a gradient where an continuously increasing ratio of organic is used.        5. A column cleaning step where all analytes of interest have been eluted but an extra high concentration of organic solvent is applied to the column in order to remove strong-binding molecules prior to the next analysis cycle. Such molecules would typically include pollutants such as organic polymers, surfactants, and very large bio-molecules that would interfere with subsequent separations if allowed to accumulate on the solid phase material.        6. A column re-equilibration step wherein the high-organic buffer inside the column is displaced with pure A-buffer such that the solid phase material can bind the next sample that is loaded.        
Steps 5 and 6, or just step 6, can also be performed as the first step instead of being the last step(s) of the cycle. The above six steps of the operation can also be classified differently and be sub-divided into yet other steps.
The flow rates may change widely from step to step and also within each step. It is possible to regulate the flow and obtain working chromatography by regulating pump speeds such that the system strives to achieve a requested back pressure. This method of “pressure regulated flow control” is unfortunate inasmuch as it can lead to highly variable and badly controlled flow rates. Hence this control method is now very rarely used, and a more common way to regulate the pump speeds is by controlling for the actual absolute flow rates as measured by flow rate sensors somewhere downstream of the pump. Hereinafter, these two different modes of pump regulation will be referred to as “pressure control” and “flow control”, respectively.
In the prior art the above steps, that encompass one analysis cycle, are either regulated by pressure control or flow control, with flow control being by far the most commonly used regulatory method. On some prior art systems, the pressure and flow during analysis execution are not regulated at all.
The detection efficiency by means of electrospray mass spectrometry is dependent on the analyte concentration at the time of ion formation, so the best sensitivity is achieved when analytes are eluted in volumes that are as small as possible.
This is done by using columns of narrow inner diameters and low flow rates. The narrow inner diameter of columns causes a large resistance to the flow of the mobile phase. Although the columns are filled with stationary phase material, the flow through the columns and the LC transfer lines is largely following Poiseulle's Law concerning the laminar flow of a Newtonian fluid in circular tubes. The Poiseulle equation states that the flow resistance is proportional to the length of the column and the viscosity of the mobile phase, while it is also inversely proportional to the column radius to its fourth potential. Hence, the resistance increases as columns become longer and narrower, especially during times of the analysis where the organic solvent ratio of the mobile phase is low. The “back-pressure” of the LC system is hereinafter considered as being the pressure delivered by the LC pumps in order to obtain a desired flow. Every LC system has an upper limit of its back-pressure above which either safety mechanisms will turn off the pumps (thereby halting the analysis) or system components will begin to fail (e.g. valves, seals, and fittings will break or leak).
In any case, it is currently necessary to take extraordinary care to ensure the back pressure does not exceed the system limitations at any point of the analysis cycle. For that reason, it is customary to perform a few “dry-runs” and monitor the back-pressure whenever a new column, or a new mobile phase, or a new method is deployed. Then the analysis parameters are typically adjusted such that the maximum back-pressure lies well below the system limits, including an extra safety margin since it is an established problem that columns exhibit increasing back-pressure contributions over time (owing to the accumulation of particulate debris at the column front and in-between column beads). The system's back-pressure limitation typically does not put any severe constraints on the execution of analyses during the part of the execution where the analytes are eluted (during the gradient), since that is typically done using very low flow rates (which in turn leads to a low back pressure).
However, in order to save considerable amounts of analysis time, it is typically advantageous to significantly increase the flow rates during the parts of the analysis cycle where: i) a sample is loaded onto a column; ii) the column or sample is being de-salted; and iii) the column is being re-equilibrated. During such parts of the analysis cycle, the system back pressure limitation poses severe constraints, and experimental parameters must (with current technology) be selected with a substantial safety margin, thereby leading to loss of analysis time. Otherwise, the execution will fail with substantial frequency, thereby leading to loss of samples and/or instrument damage and/or subsequent loss of time (since samples have to be re-analyzed).
Typical prior art nano liquid chromatography systems have upper back-pressure limits of around 5,000 PSI (340 bar), whereas some systems are designed to remain operational at over 10,000 PSI and are called Ultra-high performance liquid chromatography systems (UPLC). UPLCs are however designed to take advantage of columns that in general elevates the back pressure, so the general problem remains and one must still take active steps to avoid an “over-pressure” situation during all parts of the analysis cycle.